Diabetes is a disease derived from multiple pathogenetic factors and generally there are two types of diabetes. Patients with type I diabetes or insulin-dependent diabetes mellitus (IDDM) barely produce or cannot produce insulin which is a hormone regulating a use of carbohydrates. And patients with type II diabetes or non-insulin-dependent diabetes mellitus (NIDDM) show the same or increased plasma insulin level compared to patients with no diabetes. However, the type II diabetes patients develop a resistance to insulin-stimulated-glucose and lipid metabolism in main insulin-sensitive tissues, i.e. muscle, liver, and fat tissue. Although plasma insulin level can be increased, it is not sufficient to overcome the significant insulin resistance, thereby causing hyperglycemia. Continued or unregulated hyperglycemia is associated with increased early morbidity rate and mortality rate. Often times, abnormal increase in sugar level is directly and indirectly related to the metabolic and hemodynamic changes in the diseases associated with the metabolisms of lipid, lipoprotein, apolipoprotein, and others. For example, patients of type II diabetes mellitus especially have a high risk of developing a coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, and neuropathy as well as giant hemangioma and microvascular complications.
The currently used therapies for treating type II diabetes include administration of foreign insulin, oral administration of drug, diet therapy, and exercise therapy. In 2005, exenatide (Exendin-4: Byetta) was approved by FDA as a supplemental therapy for type II diabetes patients who could not get the appropriate glucose regulation even with taking metformin and/or sulphonylurea.
Exenatide (exendin-4) is a strong GLP-1 receptor agonist and is produced in the salivary gland of lizard. Exendin-4 shows affinity to insulin, suppress food intake and gastric emptying, and show affinity to β-cells in rodents (Parks et al., Metabolism. 50: 583-589, 2001; Aziz and Anderson, J. Nutr. 132: 990-995, 2002; and Egan et al., J. Clin. Endocrinol. Metab. 87: 1282-1290, 2002). In addition, as glycine is present at position 2 of the N-terminal of exidine-4, it is not a substrate for DPPIV unlike GLP-1. Disadvantage of using exenatide is a short half-life (t½) which is only 2 to 4 hours, and thus it has to be injected twice per day (Kolterman et al., J. Clin. Endocrinol. Metab. 88: 3082-3089, 2003 and Fineman et al., Diabetes Care. 26: 2370-2377, 2003).
Peptides like the above-described exenatide are easily denatured or degraded by proteases in the body due to low stability and lose activity. Also, the size of exenatides are relatively small and thus easily removed by the kidney. Hence, drugs containing peptides as pharmaceutically active ingredients have to be frequently administered to patients in order to maintain the target serum level and titer thereof. Mostly, the peptide drugs are administered to the patients in the form of injection and at high frequency to maintain the serum level of physiologically active peptide, but this causes a lot of pain in patients.
There have been many attempts to solve these problems, and one of them was the delivering of a peptide drug into the body through oral or nasal inhalation by increasing the biomembrane permeability of the peptide drug. However, this method has significantly low efficiency for delivering the peptide into the body compared to injections. Therefore, there are still many limitations in maintaining the activity of peptide drug in vivo at the required level.
Meanwhile, there have been continuous attempts to maximize therapeutic effects of drug by improving the stability of peptide drug in blood and maintaining a high drug level in blood for a long period of time. These long-acting formulations of peptide drugs should promote an increased stability of peptide drug and also maintain a sufficiently high titer of drug itself without inducing immune responses in patients.
As a method for stabilizing peptides and preventing peptide degradation by protease, there have been many attempts to modify a specific amino acid sequence sensitive to protease. For example, GLP-1 (7-37 or 7-36 amide) that is effective in treating type II diabetes by reducing blood glucose level has a half-life as short as below 4 minutes (Kreymann et al., 1987). The short half-life is due to loss of titer of GLP-1 through peptide cleavage between amino acid No. 8 (Ala) and No. 9 (Asp) of GLP-1 by dipeptidyl pepdidase IV (DPP IV). Thus, there have been many studies on developing GLP-1 derivatives having resistance to DPP IV, and in these studies, Ala8 was substituted by Gly (Deacon et al., 1998; Burcelin et al., 1999), or by Leu or D-Ala (Xiao et al., 2001) for increasing resistance to DPP IV while maintaining the peptide activity. Also, the N-terminal amino acid of GLP-1, His7, is an important amino acid for GLP-1 activity and also a target of DPP IV, and thus in U.S. Pat. No. 5,545,618, the N-terminal was substituted by alkyl group or acyl group. Likewise, in Gallwitz et al., His7 was N-methylated or alpha-methylated, or the whole His was substituted by imidazole for increasing peptide resistance to DPP IV while maintaining bioactivity (Baptist Gallwitz, et al., Regulatory Peptides 86, 103-111, 2000).
Besides these variants, exenatide (exendin-4, U.S. Pat. No. 5,424,686) which is a GLP-1 derivative purified from a salivary gland of glia monster has a resistance to DPP IV and a higher bioactivity than GLP-1, thereby having 2 to 4 hour-long half-life in the body which is a lot longer than that of GLP-1. However, a sufficient in vivo duration of bioactivity cannot be derived solely by increasing the peptide resistance to DPP IV. For example, the currently available exendin-4 (exenatide) has to be administered twice a day to patients through injections, which brings undue burden to the patients.
A limitation of these insulinotropic peptides is in that the size of peptide is too small to get collected in the kidney and thus it is easily lost outside of the body. Therefore, in order to prevent the loss of peptide in kidney, a highly soluble macromolecule such as polyethylene glycol (PEG) has been attached to the surface of peptide.
PEG binds to a specific site or various sites of a target peptide non-specifically and increases the molecular weight of the peptide, which then prevents the loss of peptide in kidney and hydrolysis of peptide, without causing side effects. For example, WO2006/076471 discloses that by attaching PEG to a B-type natriuretic peptide (BNP), which activates production of cGMP by binding to NPR-A and reduces intra-arterial blood pressure, thereby being effective as therapeutic agent for congestive heart failure, the bioactivity of BNP can be maintained. Likewise, U.S. Pat. No. 6,924,264 describes a method for increasing the in vivo durability of exidine-4 by attaching PEG to lysine residue of an exidine-4. However, while these methods can extend the in vivo durability of a peptide drug by increasing the PEG molecular weight, the titer of the peptide drug gets remarkably reduced as the PEG molecular weight increases, and also the PEG reactivity with the peptide is reduced, thereby reducing yield.
As another method for increasing the in vivo stability of physiologically active peptide, a method for producing a fusion protein, where the genes for peptide and physiologically active protein are linked through genetic recombination and the cells transformed with the recombinant gene are cultured, has been developed. For example, a fusion protein producing exendin-4 which is fused to transferrin (Tf) through polypeptide linker was previously reported (Korean Patent Application No. 10-2009-7003679). Also, as a method for using immunoglobulin, a fusion protein of GLP-1 derivative where GLP-1 derivative is fused to IgG4 Fc was also disclosed before (Korean Patent Application No. 10-2007-7014068).
Recently, as a long-acting protein and peptide drug formulation which can promote a minimal reduction in activity and an increased stability, a conjugate generated by combining immunoglobulin Fc region, non-peptidyl polymer, and physiologically active polypeptide is disclosed in Korean Patent Registration No. 10-0567902 (Physiologically active polypeptide conjugate having improved in vivo durability) and Korean Patent Registration No. 10-0725315 (Protein complex using an immunoglobulin fragment and method for the preparation thereof).
Through the above method, insulinotropic peptide may be applied as a physiologically active polypeptide for preparing a long-acting insulinotropic peptide conjugate (Korean Patent Registration No. 10-2008-0001479). To manufacture the drug comprising a long-acting insulinotropic peptide conjugate, it is essential to prevent physiochemical changes such as heat-induced denaturation, aggregation, adsorption, or hydrolysis caused by light, heat, or impurities in additives during storage and delivery processes while maintaining in vivo efficacy. In particular, a long-acting insulinotropic peptide conjugate has larger volume and molecular weight compared to the insulinotropic peptide itself, and thus it is hard to stabilize.
Generally, proteins and peptides have a short half-life and can undergo denaturation, such as aggregation of monomers, precipitation by aggregation, and adsorption to the surface of container, when exposed to unsuitable temperatures, water-air interface, high pressure, physical or mechanical stress, organic solvents, and microbial contamination. The denatured proteins and peptides lose their inherent physiochemical properties and physiological activity. Since protein denaturation is irreversible in most cases, the denatured proteins and peptides cannot recover their inherent properties. Also, it is likely that the proteins are unstable and easily affected by outside factors such as temperature, humidity, oxygen, ultraviolet rays, and thus they undergo physical or chemical changes including aggregation, polymerization, or oxidation, thereby losing activity.
Also, the adsorbed proteins and peptides are apt to aggregate as they denature, and when the aggregated proteins and peptides are introduced into the body, they may cause antibody formation. Thus sufficiently stable proteins and peptides must be administered. In this regard, there have been various methods developed to prevent the denaturation of protein and peptide in solution (John Geigert, J. Parenteral Sci. Tech., 43, No5, 220-224, 1989, David Wong, Pharm. Tech. Oct. 34-48, 1997, Wei Wang., Int. J. Pharm., 185, 129-188, 1999, Willem Norde, Adv. Colloid Interface Sci., 25, 267-340, 1986, Michelle et al., Int. J. Pharm. 120, 179-188, 1995).
For producing some of protein and peptide drugs, a freeze-drying process has been used to solve stability issue. However, this process is inconvenient in that freeze-dried products have to be dissolved in solvents for injection again before use, and it requires a large-scale investment such as using a large number of freeze-driers since the freeze-drying process is involved in the manufacturing process. Alternatively, powdering method using a spray drier has also been used. However this method has low economical value due to low product yield and may give negative effect on product stability since the proteins are exposed to high temperature.
As an alternative approach to resolve these limitations, other studies tried to add stabilizers to the protein and peptide in solution to prevent physiochemical changes of protein drug while maintaining in vivo efficacy thereof during long-term storage. A type of protein, human serum albumin, has been widely used as a stabilizer for various protein drugs, and the efficacy thereof has been approved (Edward Tarelli et al., Biologicals (1998) 26, 331-346).
Purification of human serum albumin involves inactivation of biological contaminants such as mycoplasma, prions, bacteria, and viruses, or screening or inspecting of one or more biological contaminants or pathogens, but even with these processes, those contaminants may not be completely removed or inactivated. Thus, patients may be exposed to these biological contaminants or pathogens when administered with human serum albumin. For example, although screening process involves the inspection of certain virus in the blood sample of donor, the inspection process is not always reliable and cannot detect certain viruses that are present in small number.
Due to their chemical differences, different proteins may be gradually inactivated at different rates under different conditions during storage. That is, the extension of storage term by a stabilizer is not the same for different proteins. For this reason, the suitable ratio, concentration, and type of stabilizers that are used to improve storage stability of proteins vary depending on the physiochemical properties of a target protein. Furthermore, when different stabilizers are used together, they may induce adverse effects different from those desired, due to competitive interaction and side effects. Also, during storage, the property of stored protein or concentration thereof can change, thereby causing different effects.
Therefore, it takes a lot of efforts and cautions to stabilize proteins in solution. Particularly, a long-acting insulinotropic peptide conjugates having improved in vivo durability and stability has a form of insulinotropic peptide, combined with immunoglobulin Fc region, and thus it has significantly different molecular weight and volume compared to general insulinotropic peptide. Therefore, a special composition is required for stabilizing the protein. Also, an insulinotropic peptide and an immunoglobulin Fc region are physiochemically different peptide or protein, and thus they have to be stabilized concurrently. However, as described above, different peptides or proteins may be gradually inactivated at different rates under different conditions during storage due to the physiochemical difference thereof. Also, when the stabilizers that are suitable for each of peptide or protein are used together, they may induce adverse effects different from desired effects, due to competitive interaction and side effects. Therefore, as for a long-acting insulinotropic peptide conjugate, it is highly difficult to find a stabilizer composition that can stabilize both an insulinotropic peptide, and an immunoglobulin Fc region concurrently.
Recently, a formulation of protein and peptide that can be used repeatedly for the patient's convenience has been developed. However, the multiple-use formulation must contain a preservative to prevent the microbial contamination after repeated administrations and prior to disposal. The multiple-use formulation containing preservative has a few advantages compared to a single-use formulation. For example, as for a single-use formulation, a large amount of drug is wasted depending on the difference in dosage. But by using a multiple-use formulation, the amount of product wasted can be reduced. Furthermore, the multiple-use formulation can be used several times without concerning about microbial growth within certain period, and since it can be supplied in a single container, packing can be minimized, leading to economic benefits.
However, use of preservative may affect the protein stability. The most well-known problem in use of preservative is precipitation issue. Precipitation of protein can reduce therapeutic effects of drug and when administered to the body it can induce unexpected immune response. Therefore, it is critical to select a type and appropriate concentration of preservative that maintain the ability to prevent microbial contamination while not affecting protein stability.